A sequence listing referring to three synthetic peptides is attached at the end of this application.
The present invention relates to peptides (and methods) for inhibiting two-component signal transduction systems, particularly sensor histidine kinase/cognate receiver aspartyl regulatory protein systems believed responsible for antibiotic resistance developed by certain pathogenic bacteria.
The two-component signal transduction network plays a important role in, among other things, the control of bacterial gene expression, particularly with respect to adaptation to environmental stress. This network includes genes that regulate antibiotic synthesis, antibiotic tolerance (e.g. vancomycin tolerance), toxin production, adaptation to physical stress, xenobiotic metabolism, environmental remediation, phytochrome-based light response, sporulation, motility, quorum sensing, biofilm formation, and virulence.
The proteins which initially mediate the control of these systems, histidine kinases and their cognate aspartyl phosphate response regulators, are also known functionally as xe2x80x9ctransmittersxe2x80x9d and xe2x80x9creceiversxe2x80x9d, respectively. Because such transmitters often sense the environment, they are also referred to as xe2x80x9csensory kinasesxe2x80x9d.
Such xe2x80x9ctwo-component signal transduction systemsxe2x80x9d are found in bacteria, yeast, fungus and plants. A general discussion of such systems is included in the monograph J. Hoch and T. Silhavy, Two-Component Signal Transduction, ASM Press, Washington, D.C. (1995). See also T. Mizuno, 123 J. Biochem. 555-563 (1998) and M. Pirrung, 6 Chem. Biol. R167-R175 (1999).
A wide variety of such transmitters and receivers have already been identified and cataloged. For example, for 34 bacterial genomes whose sequence determination is completed information relating to these transmitters and receivers is readily available in a specialized database called xe2x80x9cSENTRAxe2x80x9d (Sensory Transduction Histidine Kinases). The SENTRA web page is funded principally by United States Department of Energy.
The antibiotic resistance two-component systems of initial interest function by autophosphorylation of a transmitter sensor His kinase protein. The phosphoryl moiety is then transferred to an Asp residue of a receiver response regulator protein, symbolically indicated as Hisxe2x86x92Asp, or Hxe2x86x92D. As a consequence of such Asp phosphorylation, the receiver protein shows an increased affinity for a DNA promoter that it binds adjacent to, and thereby activates transcription of the gene(s) that the promoter regulates. In some cases the genes express proteins that ultimately are responsible for antibiotic resistance or tolerance.
The number of such transmitters and receivers in pathogenic bacteria is 26 and 33, respectively, in E. coli, but can vary from 6 and 3, respectively, in Neisseria gonorrhoeae to 56 and 76, respectively, in Pseudomonas aeruginosa. The presence of unequal numbers of receivers and transmitters in a cell indicates that some transmitters or receivers are linked to more than one cognate counterpart.
Three two-component signal transduction systems in S. pneumoniae have been identified by name as, ComDe, CiaHR, and VncRS. These systems regulate, respectively, competence for DNA-mediated transformation, resistance to cephotaxime, and vancomycin tolerance. VncRS is of special interest because vancomycin is an important antibiotic of xe2x80x9clast resortxe2x80x9d, and the extent to which bacteria develop tolerance to it diminishes the backup utility of this antibiotic.
VanR and VanS comprise the respective transcriptional response regulator protein and sensor kinase protein of a two-component signal transduction system which regulates vancomycin resistance in Enterococcus faecium. See generally A. Haldimann, 179 J. Bacteriol. 5903-5913 (1997). Transcriptional activation of what is known as VanHAXYZ gene cluster is a key event leading to the expression of resistance in induced cells.
VanS, a sensor kinase which is responsive to the presence of vancomycin in the medium, undergoes a net increase in the level of phosphorylation of one of its residues, His-164, by ATP. VanS then transfers the phosphoryl group from His-164 of VanS to Asp-55 of VanR, the response regulator for vancomycin resistance, thereby increasing its affinity for PVanH, the promoter for transcription of the VanHAXYZ gene cluster. VanRxcx9cP appears to bind cooperatively as a dimer to the DNA promoter PVanH where it recruits RNA polymerase to initiate transcription of the VanHAXYZ gene cluster. Thus, phosphorylated VanR which is not interfered with leads to greater expression of factors responsible for resistance to the antibiotic.
To date, there has been little success in inhibiting the development of antibiotic resistance in bacteria to certain antibiotics such as vancomycin. Further, the art wishes to develop other means for controlling the development of host cells.
In one aspect the invention provides a method of inhibiting the binding of a phosphorylated receiver aspartyl regulatory protein adjacent a cognate DNA promoter in a host cell. The host cell is selected from the group consisting of bacterial cells (preferably pathogenic bacterial cells such as streptococcus), yeast cells, fungi cells and plant cells, and the host cell is of the type having a two-component signal transduction system. A phosphorylatable receiver aspartyl regulatory protein from which the phosphorylated receiver aspartyl regulatory protein is derived is one component of this system.
In accordance with this method, one exposes the host cell to a peptide of less than two hundred amino acid residues (preferably less than fifty or thirty, even more preferably between six and eighteen residues), the peptide having a histidine residue and having an amino acid sequence portion of at least six amino acid residues which mimic a sensor histidine kinase in the cell that is a second component of the two-component signal transduction system. In this regard, in the absence of the peptide the kinase phosphorylates the phosphorylatable receiver aspartyl regulatory protein.
For purposes of this patent, a peptide is deemed to mimic another amino acid sequence if over a region of at least six amino acid residues of the peptide there is at least 25% (preferably at least 50%, even more preferably at least 66%) homology with respect to a sequence of amino acids of the same length within thirty (preferably within twenty) amino acid residues of a phosphorylation histidine center on the kinase. An even more preferred form of mimicking is if over a region of at least twelve amino acid residues of the peptide there are two such at least six amino acid residue regions of the peptide. One of these regions has at least 50% homology with respect to a first sequence of amino acids of the same length within twenty amino acid residues of the phosphorylation histidine center on the kinase, and another of these regions of the peptide has at least 50% homology with respect to a second sequence of amino acids of the same length within twenty amino acid residues of that phosphorylation histidine center, albeit on an opposite side of the phosphorylation histidine center than the first sequence.
In preferred aspects the host cell is a bacterial cell and the two-component signal transduction system participates in the regulation of the development of resistance of the cell to an antibiotic such as vancomycin. For example, the peptide can be selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2.
In another form the invention provides a method of inhibiting the binding of a receiver aspartyl regulatory protein to a sensor kinase in a host cell. The host cell is again selected from the group consisting of bacterial cells (preferably pathogenic bacterial cells such as streptococcus), fungi, yeast and plant cells, and the host cell has a two-component signal transduction system of which the receiver aspartyl regulatory protein is one component.
In accordance with this method one exposes the host cell to a peptide of less than two hundred amino acid residues (preferably less than fifty or thirty, even more preferably between six and eighteen residues), the peptide having a histidine residue and having a sequence of at least six amino acid residues which mimic a sensor histidine kinase in the cell that is a second component of the two-component signal transduction system. In this regard, in the absence of the peptide the kinase is involved in phosphorylating said receiver aspartyl regulatory protein.
Other preferred forms of this embodiment are peptides capable of reducing the ability of a bacterial cell to tolerate vancomycin. These could be selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2. However, they could also be other peptides that are between six and eighteen amino acid residues in length that mimic the VanS kinase.
Another aspect of the invention provides methods for identifying the above peptides. One approach is, for a selected two-component signal transduction system, to obtain a combinatorial peptide library of peptides of between six and thirty amino acid residues. Preferably, a plurality of peptides in the combinatorial peptide library have a histidine residue and an amino acid sequence portion of at least six amino acid residues which mimics a sensor histidine kinase that is a component of that two-component signal transduction system. One then selects at least one candidate peptide therefrom based on its ability to bind to a receiver aspartyl regulatory protein which is another component of that selected system.
Another approach is, for a selected two-component signal transduction system, to obtain a sequence of a sensor histidine kinase which is one component of the system, and select at least one candidate peptide which mimics that kinase.
It as been surprisingly learned that such two-component signal transduction systems can effectively be inhibited by providing a peptide ligand having less size than the natural kinase that still binds tightly to the receiver regulatory protein, and that the structure of such ligands should preferably closely mimic amino acid sequences adjacent the histidine center of the sensor protein to achieve this. This discovery should be useful across a wide range of pathogenic bacterial cells, other bacterial cells, and other cells that incorporate such two-component systems, albeit particularly with respect to providing a way of inhibiting the ability of bacterial cells to develop resistance to certain antibiotics.
These and other advantages of the present invention will become apparent after study of the following specification and claims.